Alphaviruses are an ongoing global public health threat as evidenced by the continuing emergence of Chikungunya virus in the Americas. The widespread geographic distribution of competent vector mosquito species in close proximity to immunologically nave populations has led to significant outbreaks of alphaviral disease. Clinically the disease manifestations of the alphaviruses range from severe polyarthralgia, which may last several years, to fatal encephalitis. Currently there are no effective antiviral interventions for alphavirus epidemics or individual infections. Positive-sense RNA viruses, such as the alphaviruses, require the RNA genome to function efficiently in multiple processes during infection. Throughout the viral lifecycle the alphaviral genomic RNA serves three discrete, and indispensable, functions; as a mRNA for synthesis of the replicase complex, as a template for viral RNA synthesis, and as cargo for the assembly of infectious progeny virions. Despite the recognition, and importance, of the above viral RNA functions, the molecular regulation of the viral genomic RNA function is largely uncharacterized. This proposal reports utilizes a robust discovery system by which host factors that interact with the viral genomic RNA during the infection of tissue culture cells can be identified. The goals of this proposal are two-fold; i) to identify via a novel discovery system the host factors that interact with genomes of several alphaviruses, including Sindbis, Chikungunya and Venezuelan equine encephalitis virus during infection, and ii) determine the site of binding for candidate host factors and functionally validae their role(s) in the regulation of genomic RNA function during viral infection. The findings of thi proposal will form the basis for further proposals to determine the molecular mechanisms by which the specific interactions modulate translation, replication and packaging of viral genomic RNA. In addition, the candidate factors identified as a direct result of this proposal may serve as the basis for the rational development of antiviral therapeutics and vaccine candidates.